MXPA00001185A - Refrigeration preservation, suspension culture and maturation of somatic embryos of gymnosperms - Google Patents

Abstract

The invention concerns a novel method for the production of mature somatic embryos of gymnosperms, which is based on performing refrigeration and suspension culture to preserve the embryogenic capacity of immature somatic embryos. The method involves keeping the immature somatic embryos in aliquid medium at 4 DEG C for a maximum period of one year;reinduction and proliferation of the immature embryos;performing treatment to reduce proliferation and maturation of somatic embryos. All steps except the last were carried out by means of suspension cultures. Embryo germination and plant development were carried out by conventional means.

Description

CONSERVATION IN REFRIGERATION, CULTIVATION IN SUSPENSION AND MATURATION OF SOMATIC EMBRYOS OF GYMNOSPERMS FIELD OF THE INVENTION The invention refers to a new alternative method of conserving conifer embryogenic tissue for long periods without the need for cryopreservation (-196 ° C), which is very important to maintain the capacity of immature embryos to a reduced cost during the time required for evaluations of each of the genotypes; It should be noted that cryopreservation is a high cost method that can produce somaclonal variation. In addition, somatic embryos that come from suspension cultures can not be cryopreserved. This new method can be used in programs of genetic improvement, genetic transformation or any other activity that requires to conserve the embryogenic tissues with all their characteristics and qualities for long periods, that is to say that the reinduction of the embryo multiplication can be achieved and later its maturation and development to plants.

BACKGROUND OF THE INVENTION The regeneration of plants by in vitro culture is based on the totipotentiality of somatic cells, proposed by Haberlandt that was published in 1902, which means that by placing the cells in appropriate conditions and stimuli can regenerate whole plants (Tautorus etal., 1991). The in vitro micropropagation systems are meristem culture, organogenesis and somatic embryogenesis; in the latter, a somatic cell or a group of them originate complete bipolar structures called somatic embryos, analogous to zygotic embryos (Ammirato, 1983). Countries such as Canada and Sweden with high technology of forest management and exploitation, have implemented strategies for sustainable use through conventional propagation and in vitro, together with the improvement of tree species to increase genetic gain and decrease the physiological turn (Gupta et al. , 1993). Currently, plants obtained from Picea abies by Hogberg et al. (1998) where they obtained 2519 embryos belonging to 12 families with a conversion to plants of only 25%. Likewise, in Pinus strobus an analysis of 52 genotypes belonging to 13 families was made, 800 mature somatic embryos were obtained in 30 genotypes of 12 families, and a conversion to plants of only 1% (Garín et al., 1998). Recently it has been reported by Ramirez-Serrano and collaborators (1999a, 1999b), the creation of a method for P? Nus sylvestris that made possible the production of somatic embryos in 70 of 82 tested genotypes, belonging to 20 families that make the use feasible of this technology for genetic improvement. The work to obtain suspensions of conifers was initiated by Durzan and collaborators, who since 1968 joined in order to achieve somatic embryogenesis in conifers; they were able to observe the patterns of development, metabolism and growth of different types of cells, which served to characterize callus and cell suspensions of conifers; however, they observed what is now known as spheroblasts (Tautorus et al., 1991). It was up to 17 years later, when the induction of somatic embryos in a broadleaf was achieved for the first time. { Liquidambar styraciflua) by Sommer and Brown (1980), this work was the basis for obtaining somatic embryogenesis in conifers. Three groups of researchers induced what is known as embryogenic tissue, embryogenic callus or clusters of embryonic suspensors characteristic of conifers (Hakman et al., 1985; Chalupa, 1985; Nagmani and Bonga, 1985); all described a white, mucilaginous and translucent tissue that, seen under the microscope, presented long suspensory cells, linked to dense meristematic cells of the embryonic apex. That year the first report of conifer regeneration was held. { Picea abies) by Hakman and von Arnold (1985). Zygotic embryogenesis has been described in almost all genera of conifers by the basic cell biology of seed germination (Owens and Blake, L985); This process is presented in two phases: the pre-embryo within the archegonium and the elongation of the latter within the gametophyte, the latter is almost always divided into several stages, in the majority of the conifers the simple or archaegonial polyembrogenesis is presented which is the fertilization of more than one egg by different pollen grains inside an egg, resulting in different genotypes inside the same seed. It also has poliembriogenesis by cleft, which begins when the base of the embryo is divided into four different vertical series of cells, so that each one forms an independent and genetically identical embryo (Owens and Melder, 1984). In Woods' works published in 1953, it was pointed out that for zygotic polyembrogenesis to occur, the availability of nutrients is more important than the genetic factor (Tautorus et al., 1991). On the other hand, it is presumed that the origin of somatic embryos is a cell or small cellular aggregates that by means of an asymmetric division form the embryonic apex and the suspensory cells; the possible development of small meristematic cells within the suspensor is also suggested, or by a mechanism similar to that of polyembryogenesis by cleft that occurs in the cells of the head of the embryo (Hakman et al, 1987). In Picea abies the embryogenic tissue begins in the hypocotyl and something similar in the radicle, but the latter did not regenerate embryos (Becwar et al., 1988), in contrast to what was found by other researchers in explants of the genus Pimis (Gupta and Durzan , 1986). It has been reported that the somatic embryos of Pinus taeda are monozygotic because it is only the cloning of an embryo, whose clones germinated and were stored as synthetic seed (Gupta and Durzan, 1987). However, according to Becwar et al. (1991) it is possible to find multiple genotypes in embryogenic tissue of Pinus taeda, from a single seed. For Picea glauca comlex. engelmannii, by means of isozyme analysis, non-significant somaclonal variation was found among subclones of a genotype, thus ensuring a massive propagation with genetic stability of a desirable genotype (Eastman et al, 1991). It is important to know how the maturation process of the seeds is carried out. It is known that the greatest amount of substances accumulates in the cells during the development of the seed in the form of proteins and lipids (Ching, 1963); bromatological analyzes of stored pine seeds have been performed, where the total protein content is between 10 and 25% of the total weight, most of these are insoluble and 80% make up the gametophyte (Gifford, 1988). In the normal development of a zygotic embryo, endogenous ABA was found to induce the accumulation and storage of reserve substances such as the aforementioned proteins, as well as fats and carbohydrates (Karssen et al., 1983). And while the humidity conditions do not change, the protease inhibitors will protect the cells until the hydrolysis that precedes germination starts (Salmia, 1980). In somatic embryos it was found that the proteins are hydrolyzed in the gametophyte and in the embryo before germinating, in such a way that they become nutrients for the plant in its first stage of development (Hakman et al, 1990). It was found that if the maturation process is carried in petri dishes, these nutrients are found in a lower concentration than zygotic embryos, and affect germination rates (Attree and Fowke, 1993); but, when maturing them in bioreactor, said substances are in equal or greater concentration than in zygotic embryos (Attree et al, 1994). Cryopreservation has basically been used as the maintenance method for embryos or cells of different species including animal structures. However, it has been proven that in conifers there is what is known as somaclonal variation which means that in mature genotypes there may be alterations in maturation and germination., although this variation has only been detected in some embryogenic crops without being detected in trees that regenerate (De Verno et al, 1998). It has been proven that this method maintains the totipotentiality of the cells, through the regeneration of plants starting from protoplasts from cryopreserved embryos of Picea glauca (Attree et al., 1989). The reference is that it is possible to maintain mature seeds for long periods at 4 ° C to evaluate their embryogenic potential. This was done to evaluate the species that produce seed in low quantity or is not regularly counted with the type of plant material to initiate the protocols of somatic embryogenesis. It was determined in mature zygotic embryos of Picea morning and P. glauca, that 38 and 18% of embryogenic tissue can be obtained after remaining 4 to 10 years, respectively (Attree et al, 1989); This has been confirmed with seeds of P. glauca stored for 11 years, where it was possible that 40% of them were stimulated to form somatic embryos (Tremblay, 1990). Mature and immature zygotic embryos, female gametophyte, and parts of seedlings developed in vitro are also used, depending on the protocols already developed (Tautorus et al., 1991, Aitken-Christie and Connett, 1992). However, the components of the medium are the determinants, for example the ratio of the sources of inorganic nitrogen and the carbon source are the key in the induction of somatic embryos (von Arnold, 1987). A high concentration of auxins and fewer cytokinins are also required in the medium (Hakman and von Arnold, 1985); however, it has been shown that for the Abies genus only cytokinins are necessary (Norgaard and Krogstrup, 1991). Ramirez-Serrano et al. (1999a) reported that a high ratio of ammonium to nitrate favors maturation and that the 9: 1 ratio produces a large number of somatic embryos.

For multiplication, the same induction medium is used with the same supplements, in solid or liquid, it should be noted that in suspension cultures, embryos multiply more rapidly and the medium is more economical (Becwar et al, 1988; Christie and Connett, 1992; Dunstan et al, 1995; Gupta et al, 1993). To maintain the embryogenic capacity, the cryopreservation of immature somatic embryos is used, which is a way to maintain important genetic resources, since they can be preserved for a long time without changing their potential (Tautorus et al, 1991). Another way to conserve in the short term is placing the immature embryos in solid medium in Erlenmeyer flasks covered with wax covers, where they can remain without subculture up to one year (Joy et al, 1991). Ramírez-Serrano (1996), found that embryos can be stored in liquid medium at 4 ° C without losing their proliferation capacity, however they could not regenerate plants. For the maturation process, a pretreatment is generally used to improve the response to the maturation medium, through the action of activated carbon, by absorbing substances such as ethylene and growth regulators that in excess can affect the ripening process (George, 1993) . Generally the same means of multiplication is used, preferably in solid, where the assimilable source of carbon, the racemic mixture of ABA, PEG or high concentration of sugars is added to make negative the osmotic potential of the medium (Attree and Fowke, 1993). . Dunstan and colleagues (1993) suggest using between 36 and 60 micromoles of ABA to have synchronization and high percentages of maturation and germination. A synergistic effect has been reported between BA and ABA, since they mature 10 times more embryos when treated with BA and then exposed to ABA, than when they are not treated with BA (Bozhkov et al, 1992). Attree et al. (1994) obtained a large quantity of somatic embryos of Picea glauca in bioreactor, of excellent quality and at very low cost. It has been reported that with the use of 1% "gellan gum" without adding PEG to the maturing medium, mature somatic embryos are obtained in different pine species (Klimazsewska and Smith, 1998, Lelu et al., 1999). center of diversity of the genus Pinus, where 50% of the known species in the world are found (McVaugh, 1994). Thousands of hectares are cut daily to meet industry demands, and although Mexico has eminently forestry characteristics, adequate forest exploitation systems are not used because the best specimens are harvested for sawing and not as seed producers for afforestation (Ramírez -Serrano ^ 1992). Genetic improvement is one of the goals of somatic embryogenesis. For tree species, multiplication of clones is considered a type of improvement, in addition to increasing productivity (Bonga, 1987), and a high genetic gain can be obtained (Gupta et al, 1993). It is also used to generate large quantities of synthetic seed in low production species; to clone varieties resistant to pesticides, pests and environmental stress; as an alternative for the conservation of germplasm of rare or threatened species and for the propagation of ornamental varieties (Attree and Fowke, 1993). On the other hand, for the processes of escalation and genetic transformation by biobalistic, it is essential to maintain the embryogenic capacity of the suspension cultures of the conifers; of Picea glauca there are transformed and stable plants with the plasmid pTVBT41100 (Ellis et al, 1993), or ABA promoters (Dc8) from the carrot in immature embryos of Picea abies, where the inserted gene has shown high levels of expression (Newton et al, 1992). Walter et al. (1999) showed that their group has transformed Pinus radiata and Picea abies with genes of economic importance. It should be noted that in Mexico, the only antecedent of somatic embryogenesis in conifers is with Pinus maximartinezii; where 2.25% embryogenic tissue was induced corresponding to 18 genotypes, immature embryos of 50 embryos / ml were multiplied to 700-1500 embryos / ml in 7-15 days depending on the genotype; however, only aberrant embryos could mature (Ramírez-Serrano, 1996).

BRIEF DESCRIPTION OF THE INVENTION According to the present invention, a new method for preserving the embryogenic capacity by suspension culture is presented, and that allows the production of mature somatic embryos of gymnosperm from immature embryos that were conserved in refrigeration. This method is characterized by giving the immature somatic embryo that originated the mature somatic embryos a treatment at 4 ° C in liquid medium up to 11 months. Subsequently, having had the embryogenic tissue reintroduction in liquid medium with the lowest concentration of growth regulators tested. There was also the establishment and continuous proliferation of immature somatic embryos having proliferated in basal modifications continuously. There was also a decrease in the proliferation of immature somatic embryos, having proliferated in a liquid medium with an ammonium to nitrous ratio of 10:90 and a source of assimilable carbon. Finally, having as part of the maturation process a maturation pre-treatment with an absorbent that allows the initiation of the development of the somatic embryo prior to exposure to the maturation medium with a high nitrate content, a source of assimilable carbon, a promoter of maturation and a drying agent. This method is characterized by the conservation in a liquid medium at 4 ° C of somatic embryos of gymnosperms. For this stage, a liquid culture medium without growth regulators was used, supplemented with a source of assimilable carbon and two sources of organic nitrogen. This method allows the embryogenic capacity of immature somatic embryos to be conserved for one year. Therefore, with the present invention, it uses refrigeration as an alternative to maintain the embryogenic capacity of immature somatic embryos without special requirements, such as the cryopreservation equipment and techniques known up to now. The embryogenic tissue reinduction stage is called the stage that somatic embryos need at 4 ° C to start again the proliferation in liquid medium with a low concentration of growth regulators, which was one of the requirements to maintain adequate the proliferation of somatic embryos. The stage establishment and continuous proliferation of immature somatic embryos, is denominated in this method, when keeping in cultures in suspension the proliferation of the genotypes that were conserved at 4 ° C, in suitable levels; by using the minimum indispensable quantity of inoculum and the lowest proven concentration of growth regulators, demonstrating a proliferation without changes for more than a year. When necessary, the ammonium / nitrate ratio was modified. The method of invention also comprises the reduction of the proliferation of immature somatic embryos in suspension culture, to improve the response during the maturation process, which consists of subculturing in a liquid medium supplemented with a low ratio of ammonium to nitrate and without growth regulators. With this treatment it is assumed that the embryos, by changing the metabolic route of the use of nitrogen, proliferation becomes difficult, which allows the action of the maturation promoters. This method also includes the maturation process that constitutes the stage of the beginning of the development of the embryo. The embryo masses should be washed at least 3 times prior to transfer on a filter paper. It should be applied in solid medium with an absorbent in medium with ammonium / nitrate ratio 10:90, carbon source and without growth regulators, whose period of stay in this type of medium is indicated by immature embryos when showing the elongation of the suspension cells and bulking of embryonic heads, which was considered as the signal to transfer them immediately to medium maturation. This medium contains a high nitrate content (ratio 10-90), a carbon source, a maturation promoter and a desiccating agent, whose period of exposure is sufficient for each somatic embryo produced to develop the cotyledons and is ready the latency stage or (desiccation that does not include this method). The said somatic gymnosperm embryos produced according to the present invention include somatic embryos of conifers. The present invention has the advantage of maintaining the embryogenic capacity of valuable genotypes by a simple and inexpensive technique, at least for the evaluation stage, because no dangerous substances or sophisticated equipment are required. The present invention constitutes a special advance in the investigation of somatic embryogenesis in conifers, especially for pináceas, in the aspect of a wide range of genotypes that can be conserved by this technique including cryopreserved genotypes. It was proved that each genotype requires different time for the reinduction of proliferation. It is possible to work with a wide range of genotypes to evaluate their capacity in ex vitro conditions, or their response to genetic transformation protocols by means of biobalistics without the use of expensive techniques or equipment.

DESCRIPTION OF THE DRAWINGS Figure represents the way to keep immature embryos refrigerated at 4 ° C in liquid medium. Figure Ib represents the influence of the storage time at 4 ° C in the reinduction of the proliferation of immature somatic embryos.

Figure 2a represents the influence of subculture on the number of embryos / ml that proliferate at different concentrations of growth regulators. Figure 2b shows how to reduce the proliferation of genotypes with the use of different ratios of ammonium to nitrate. It is observed that the lowest rate of proliferation was the ratio of ammonium to nitrate 10:90. Figure 3 a shows a high frequency of immature somatic embryos in suspension and that the amount of the genotype depends. The medium of better proliferation is supplemented with the ratio of growth regulators of 0.5 mg / l of 2,4-D and 0.25 of BA. Figure 3b shows an immature somatic embryo in liquid proliferation medium supplemented with the ratio of growth regulators of 0.5 mg / L of 2,4-D and 0.25 of BA. Figure 4a shows a high frequency of mature somatic embryos in a maturing medium with ammonium / nitrate ratio 10:90, 3% maltose 80 uM ABA, and 0.35% "gellan gum". Figure 4b shows a mature somatic embryo in the anterior medium. Figure 4c shows the root development of pine plants DETAILED DESCRIPTION OF THE INVENTION According to the present invention, there is a new method to regenerate plants in gymnosperms, from the conservation of immature embryos at 4o C from previously induced or cryopreserved genotypes, in any known liquid medium in the state of the technique of somatic embryogenesis in conifers, for an average period of one year, also using suspension culture in all stages except for the maturation process. The method includes the reinduction of embryogenic tissue for 3 months, establishment and continuous proliferation of immature embryos, decreased proliferation of immature embryos to improve response to maturation, maturation process that includes the start of embryo development and the maturation in solid medium. Depending on the stage, the liquid culture media were supplemented with special ratios of ammonium to nitrate, with or without growth regulators and assimilable carbon source. The present invention requires the understanding and control of certain biological factors that affect the latency, induction, proliferation and maturation of somatic embryos, because it is considered that the stages of development of the somatic embryo are similar to that of the zygotic embryo. As well as the effect of growth regulators on proliferation by forcing each embryo to originate a new one by embryogenesis by cleft and the ratio of ammonium / nitrate throughout the process of somatic embryogenesis in conifers. The zygotic gymnosperm embryos, after fertilization, develop from a non-nuclear structure, whose process may have variations. In the case of the pines the embryos have fertilization in the summer, and the development of the embryo can occur in two consecutive summers (depends on the species). A structure is formed within the archegonium with 16 cells that lengthen to form the pre-embryo that can originate the same and initiate in a natural way the poliembriogenesis by cleft of a single genotype or multiple, when more than one ovule is fertilized. If a normal process is followed, the suspensor pushes the embryonic head towards the gametophyte and begins the maturation, transferring the suspensors to the embryonic cells the nutrients they need from the base of the gametophyte, simultaneously initiating the process of desiccation of the gametophyte and the embryo. such a way that when the latter is completely mature, the humidity conditions are the minimum for the embryo to enter the dormancy stage and to remain in a latency stage until the conditions are propitious for germination and follow the normal growth to the plant. Considering the above, somatic embryogenesis in gymnosperms requires the use of the same conditions in order to carry out the normal embryogenesis process. Therefore, it is assumed that the requirements for induction, proliferation and maturation are basically the same for all conifers, with their variants for the species studied, in this case Pinus spp. These methods are characterized because they are generally different from those used for angiosperms. . For some pineapples, as in this case, it is necessary to maintain the production capacity of somatic embryos for long periods, however it is well known that with prolonged subculture the embryos lose not only the ability to mature but may lose the capacity for proliferation. In natural conditions, that is to say, with the protection of the cone and the gametophyte, a large percentage of pine embryos resist the winter or the period of low latency. Therefore, the conservation in refrigeration of immature embryos j >was evaluatedAt the same time, 5 concentrations of growth regulators were evaluated in the proliferation of Genotype 1. The basic concentration was 2 mg / l of 2,4-D and 1 mg / l of BA (100%). ), this experiment was the one that gave the pattern to use the medium for reinduction- 500 ml bottles were used with 100 ml of medium with an ammonium / nitrate ratio 40/60 without regulators and 33 ml suspension of embryogenic tissue (5, 6, 11 and 15 somatic embryos / ml) and were taken to refrigeration at 4o C. Six tests were made of the reinduction of the multiplication at different time of permanence at that temperature and different concentrations of growth regulators were evaluated in it. liquid medium. During the lo., 2nd. and 4th. months this medium was used with 100% of the CR concentration. At 6 months the reinduction was evaluated with three concentrations: 100, 75 and 50% of growth regulators, and at 8 and 11 months only with 50%. 5 ml of the embryo suspension in refrigeration was taken for each of the four repetitions per treatment. The best concentration was determined by measuring the anatomical characteristics of the embryos in proliferation, as well as the concentration of embryos per milliliter at the beginning and after 10 subcultures. After 11 months of storage and after 6 reinduction tests, the embryos had the ability to multiply; It was found that the storage time and reactivation time have a correlation of 0.94 with 99% reliability, with more storage time, longer time to activate the multiplication. For example, for 1 month they were reactivated in 3 weeks and in the last experiment performed at 11 months they took around 11 weeks. They were evaluated only until 11 months because the samples were not enough to be able to carry out further tests (Fig 1). This trial showed differences in the embryo multiplication index due to the concentration of regulators showing a positive correlation, that is to say a higher concentration of regulators higher multiplication index, not in the multiplication time where it was the same for all. Additionally it was observed that in the 2 higher levels of regulators after 2 subcultures the deformation started and where they had initially 5 and 6 embryos / ml they did not multiply in any treatment; Furthermore, in the smallest amount of regulators (50%) the multiplication was slower (50 somatic embryos / ml) but after 10 subcultures it reached the levels of 600 somatic embryos / ml, observing the embryos in perfect condition (Fig. 2a and Fig. 2a). 3b). Regarding the effect of the concentration of the growth regulators, we have the following: with the 100% concentration in the subcultures after the reinduction in months 1, 2 and 4 there was an accelerated loss of the embryogenic capacity; in month 6 it could be observed that also in the 75% concentration there was a drastic change in the characteristics of the embryos until disappearing to the 4 subcultures, not so with the 50% concentration; later, in the analysis at 8 months it was possible to observe during 10 subcultures that the quantity used and the concentration were the determinants to maintain the embryogenic capacity, being the best in the concentration at 25%, in the analysis of variance, significant differences were detected due to the effect of the regulators both on the size of the two structures of the embryos and on the total length (Table 1), finding significant differences by the LSD procedure (Table 2); and that was later verified in the evaluation carried out after the reinduction of month 11 where the embryos multiplied indefinitely every 7-15 days (Fig. 3b).

Table 1. Analysis of variance to evaluate the effect of growth regulators on the size of the somatic embryos of Genotype 1.

Table 2. Differences by growth regulators in Genotype 1. SUSPENSION HEADS COMPLETE EMBRYO DMS = 0.034 DMS = 0.1722 DMS = 0.1875 Medium Group N R (: c irupo Medium N RC C Medium Group N RC z%%% 0.24 50 25 | A 1.27 50 25 | A 1.51 50 25 ~ | B | A 0.22 50 75 B 1.09 50 100 B 1.28 50 100 -1c 0.19 50 100 B 1.06 50 50 B 1.25 50 75 lie 0.16 50 50 B 1.03 50 75 B 1.22 50 50 JD 0.14 50 0 I | C 0.76 50 0 I 0.90 50 0 DMS = Significant minimum difference; z = level of significance 0.05 Subsequently, the evaluation of storage in refrigeration and the reinduction of multiplication of several genotypes was carried out. Four 125 ml flasks were placed with 45 ml of 40:60 ammonium / nitrate concentration medium without regulators and inoculated with 10 ml of embryogenic tissue suspension of 8 genotypes to be analyzed (approximately 20 embryos / ml were suspended. in each flask). Tests of the reinduction of the multiplication were carried out after one month of refrigeration in a culture medium with a 50% concentration of growth regulators tested in genotype 1, taking 5 ml of inoculum for each repetition. For each genotype, the reinduction time and the number of embryos per ml were taken. They multiplied in the middle with the same ammonium / nitrate ratio 40:60 and concentration of growth regulators (equal to the induction medium) corresponding to each genotype (Table 3), where differences were found in the number of embryos / ml of each genotype and also in the time of his cultivation. Induction data are not shown because they have no relevance in this method, however they are shown to have proliferated in liquid in the same medium where they were induced to verify that proliferation is not maintained in the medium where embryogenic tissue is induced.

Table 3. Response to the means of multiplication of induced genotypes.

The genotypes G2, G3 and G4 proliferated in medium with the concentration of 100% regulators; G5, G6, G7 and G8 in the culture medium with 75% concentration and G9 with 50% concentration. The evaluation of refrigerated storage at 4o C and the reinduction of Multiplication. Differences in storage resistance and response time were observed by genotype, Table 4; after 1 month in refrigeration, three genotypes did not multiply, finding that others had the same behavior of the genotype Gl, however two genotypes started the multiplication until after 5 months.

Table 4. Genotypic response to storage.

Multiplication after the reinduction of all genotypes. The amount of embryos to start each subculture was decisive to use between 20-40 embryos / ml to start, since they could be left until they had 1000-1500 embryos / ml. Differences between genotypes in embryogenic or multiplication capacity were observed. They could be subcultured without problems using those amounts between 5 and 15 subcultures depending on the reinduction time.

Table 5. Response of Genotypes to Multiplication in medium with 25% of CR.

Direct multiplication after induction without storage at 4 °.

Therefore, it is demonstrated that the levels of proliferation with liquid medium are influenced by the concentration of growth regulators and the capacity of each genotype. This allowed to have for a year the continuous proliferation of the genotypes established without any malignancy or no proliferation (Table 5). It should be mentioned that the 80:20 ammonium nitrate ratio was shown to promote a high rate of proliferation in solid medium (Fig. 2b), and also in suspension cultures, preferably using the lowest concentration of growth regulators and a source of carbon that can be sucrose or maltose. On the other handIt was very important to consider an intermediate stage between proliferation in liquid medium and the maturation process, to prepare the immature embryo for the action of a maturation promoter. This capacity is acquired by immature embryos that have proliferated in a medium with ratios of ammonium / nitrate 10:90 or 20:80 (Fig. 2b), maltose at 3% and without growth regulators. For this stage it is convenient to eliminate all the previous means of proliferation, wash twice with sterile distilled water and resuspend in liquid medium. The suspension of embryos after two subcultures becomes dark, which means that the embryos are ready to transfer them to medium with an absorbent to completely stop the proliferation and / or to favor the start of the development of the embryo. To initiate the maturation process, which includes the pre-treatment of maturation in a medium supplemented with 1% activated carbon, ratio of ammonium to nitrate 10:90, 3% of maltose and gelled with 3.5% of "gellan gum", should be place between 150-200 mg per repetition, preferably as a thin layer, prior to this should be washed three times with sterile distilled water or any liquid medium with minerals to eliminate substances that induce proliferation, transfer them to the solid medium, eliminate the Excess liquid and aerate the embryos and the medium previously to remove moisture, give this treatment until the tissue shows no signs of proliferation, (2-8 weeks). In this way the response was optimized and it was possible to mature embryos from suspensions that come from genotypes kept in refrigeration at 4 ° C (Fig. 4a and Fig. 4b). For the maturation process, the interaction between the nitrogen and the carbon source and the type of carbon, combined with a high concentration of ABA, and at least one drying agent is very important. The abscisic acid must be added at the beginning of the maturation at high concentration to obtain embryos of the best quality and avoid early germination, for most conifers the concentration varies between 16 uM and 24 uM (+) ABA. However, for the Pinus genus it is required to use between 60 and 100 uM of ABA. Generally the racemic mixture (±) is the most used and has given better results, in this experiment only 80 uM ABA was used, since in previous evaluations were tested 20, 35 and 60 that showed no effect in the aforementioned lines. Embryos belonging to the orthodox type require desiccation to have a normal germination. In order to produce somatic gymnosperm embryos that do not present early germination, it is also necessary that they develop in a medium with a high concentration of a desiccating agent. Some sugars have been used as desiccant agents in a concentration of 6 to 9%, but for this new method an inert agent was used that is not assimilated by the cell, as is the case of sugars, and that produces drought conditions. which favors the accumulation of reserve substances inside the cells. When this agent is used in inadequate concentration, the embryogenic tissue either proliferates or dehydrates (Table 6). The most recommended is the PEG with molecular weight of 4000, since it gives a medium viscosity. The "gellan gum" can also be used. This component is a very important polymer in the medium, because it does not react with any substance of the medium, it is practically inert, it only serves as a support, to give the physical consistency required by a medium for in vitro culture. It is important that the medium remains without liquefying, and that it is not absorbed by the plant. Depends on its concentration the availability of water in the middle. The amount of embryos exposed to the maturation medium was decisive to optimize the method to produce the somatic embryos, and this knowledge was obtained from the genotypes with slow proliferation, by exposing to the maturation medium the available embryogenic tissue that allowed only to form a thin layer of embryos, which produced mature somatic embryos. Conversely, genotypes that have high rates of proliferation, the response to the maturation medium is practically nil. Two experiments were carried out to test the effect of one or two drying agents in the medium, using immature somatic embryos of genotypes conserved at 4 ° C. Although the concentration of "gellan gum" at 1% without PEG has been reported as optimal, but in another relation of ammonium to nitrate and other supplements- To prove the above, an experiment was carried out with a medium supplemented with the ratio ammonium / nitrate 40:60, 3% sucrose, 35 uM ABA, with or without 7.5% PEG 4000 and different concentrations of "gellan gum" (Table 6); where the embryogenic tissue is completely dehydrated in concentrations from 0.7%, and in 0.35% it was observed that the proliferation continued. However, based on the above information, a medium supplemented with ammonium / nitrate ratio 10:90, 80 uM ABA, 3% maltose, using 0.55% "gellan gum" was used to give drought conditions in the medium without PEG 4000 obtained the maturation of somatic embryos (Fig. 4a and Fig. 4b). Therefore, it was proved that it is possible to obtain mature somatic embryos by means of conservation at 4 ° C.

TABLE 6. Influence of PEG and "gellan gum" on the maturation of embryos in medium ammonium / nitrate ratio of 40:60, 35 uM ABA and 3% sucrose, using more than 400 mg of embryogenic tissue per repetition. Response Concentration "gellan gum" somatic embryos ± PEG 7.5% Mature / repetition 0.35% Proliferation 0 0.55% * Maturation 5OM0 0.7% Dehydration 0 1.0% Dehydration 0 1.4% Dehydration 0 * This treatment was given with maturation medium supplemented with the ratio ammonium to 10:90 nitrate, 3% maltose, 80uM ABA without PEG and 150-200 mg of immature embryos per repetition.

This invention has several important features. This is the first time that a method of conservation of immature gymnosperm embryos different from cryopreservation has been reported, which allows the regeneration of mature coniferous embryos (Fig. 4c). This method allows keeping the coniferous somatic embryos for a proven period of 11 months without losing their capacity for proliferation. It was shown that it depends on the genotype and the concentration of growth regulators. However, in valuable genotypes it can be reinducted when proliferation is needed and at the same time retaining the suspension for another period again at 4 ° C. This is the first time that a medium with a ratio of ammonium to nitrate 10:90 has been used in suspension cultures to reduce the proliferation of embryogenic tissue prior to the pre-treatment of maturation. It was found that the method of maturation that includes, reduction of proliferation in medium with ammonium / nitrate ratio 10:90, washing the immature embryos with sterile distilled water, using only 150 to 200 mg of the embryogenic tissue by repetition, distributed in a thin layer, pretreatment to initiate the development of the embryo in the specified medium, and exposure of the somatic embryos in the maturation medium with the ammonium / nitrate ratio 10:90, 3% maltose, 80uM ABA and 5.5% "gellan gum". It worked to mature somatic embryos that come from cultures in suspension, which were preserved in refrigeration at 4 ° C.

This methodology can be used to maintain the capacity of genotypes under evaluation for subsequent cryoconservation in conventional breeding programs and / or genetic transformation, among other studies that only successful systems of plant regeneration allow.

Definitions The term "suspension culture" is defined as the semicontinuous culture method in Erlenmeyer flasks, where immature embryos proliferate exponentially, which makes it necessary to periodically restart the crop taking the minimum amount of inoculum (immature embryos in suspension) - from the previous culture ^ add sterile liquid medium remove the culture medium, add new and take advantage of or eliminate the excess of embryos.

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Claims (41)

1. A method to produce mature somatic embryos of gymnosperms from genotypes stored in refrigerated liquid medium; comprising the 4 ° conservation technique, embryogenic tissue reinduction for 6 months in suspension cultures, establishment and continuous proliferation in suspension culture, treatment to reduce the proliferation rate in liquid medium, treatment for embryo maturation immature somatic.

2. A method according to claim 1, wherein the conservation technique at 4 ° C of immature somatic embryos comprises supplements of the preservation medium, the size of the container, the number of somatic embryos / ml and the volume of medium used.

3. A method according to claim 2, wherein the supplements are 3-5% sucrose, 250-500 mg glutamine and 500-1000 mg cazein hydrolyzate.

4. A method according to claim 2, wherein the container size is from 125 ml to 1000 ml.

5. A method according to claim 2, wherein the amount of embryos per ml is from 5 to 50 / ml.

6. A method according to claim 2, wherein the volume is from 0J to 0.6 of the capacity of the container.

7. A method according to claim 1, wherein the reinduction consists of the exposure or treatment of immature somatic embryos in a liquid medium with different ammonium / nitrate ratios, different concentration of growth regulators but in the same ratio (2). : 1) and carbon sources.

8. A method according to claim 7, wherein the liquid medium for reinduction is supplemented with different ammonium / nitrate ratios between 90:10 to 10:90.

9. A method according to claim 7, wherein the medium with different concentration of growth regulators is supplemented with 2: 1, 1.5: 0.75, 1: 0.5, 0.5: 0.25 and 0: 0 mg / l of 2.4 -D and BA respectively.

10. A method according to claim 7, wherein carbon sources are said to sucrose or maltose in a concentration of 1 to 4%.

1. A method according to claim 7, wherein the suspension is transferred each subculture to another fresh medium containing the lowest concentration of growth regulators tested.

12. A method according to claim 11, wherein the lowest concentration of regulators tested is 0.5 mg / l of 2,4-dichlorophenoxyacetic acid and 0.25 of Benzyladenine (2: 1).

13. A method according to claim 7, wherein the named embryogenic tissue is established and proliferates for at least 12 months by subculturing every 1-2 weeks in a fresh medium containing the lowest concentration of growth regulators tested.

14. A method according to claim 13, wherein the lowest concentration of regulators tested is 0.5 mg / l of 2,4-dichlorophenoxyacetic acid and 0.25 of benzyladenine (2: 1).

15. A method according to claim 1, wherein the treatment for reducing the proliferation rate in liquid medium, is the subculture with a medium supplemented with the ratio of ammonium / nitrate between 1: 90 to 30:70 and a source of carbon, one month before starting the treatment to initiate the development of the immature somatic embryo.

16. A method according to claim 15, wherein the carbon source is maltose.

17. A method according to claim 1, wherein the maturation treatment includes a treatment to initiate the development of the immature somatic embryo, in a medium with an ammonium / nitrate ratio between 10 90 to 30:70, plus an absorbent chemical, carbon source and without growth regulators for a period between 4-8 weeks.

18. A method according to claim 17, wherein it is called a treatment to initiate the development of the somatic embryo includes washing at least three times the embryogenic tissue with sterile distilled water prior to placing it in a paper filter on the medium with chemical absorbent.

19. A method according to claim 17, wherein the so-called treatment to initiate the development of the somatic embryo includes transferring between 150-200 mg of embryogenic tissue in such a way that a thin layer without clumps remains.

20. A method according to claim 17, where it is called a carbon source to maltose.

21. A method according to claim 17, wherein it is called a chemical absorbent to activated carbon.

22. A method according to claim 1, wherein maturation of the somatic embryos is referred to the development process of the embryonic head to form cotyledons in medium with the low ratio of ammonium to nitrate, carbon source, high concentration of ABA and a drying agent

23. A method according to claim 1, wherein maturation of somatic embryos is referred to as originating from genotypes conserved at 4 ° C for a period of 1-12 months.

24. A mature somatic gymnosperm embryo characterized by having been preserved at 4 ° C the immature embryo that originated it, its product after reinduction, named embryogenic tissue having proliferated adequately at the lowest concentration of growth regulators tested, having been treated for a period of time that allows to lower the proliferation to the minimum level corresponding to that genotype, to have had a treatment with an absorbent that allows the start of the development of the somatic embryo and to have had a maturation treatment in medium with a high content of nitrate, carbon source, high concentration of a maturation promoter, and a drying agent.

25. A mature somatic gymnosperm embryo according to claim 24, characterized by being analogous to a gymnosperm zygotic embryo.

26. A mature somatic gymnosperm embryo according to claim 25, wherein a conifera is named gymnosperm.

27. A mature coniferous somatic embryo according to claim 26, where an embryo is named that comes from the Pinaceae family.

28. A mature coniferous somatic embryo according to claim 27, where an embryo is named that comes from the genus Pinus.

29. A mature somatic pinacea embryo according to claim 28, wherein all those that come from the genus Pinus are named embryos.

30. A method according to claim 24, wherein the so-called embryos are developed in a time range between 1-12 weeks.

31. A method according to claim 24, wherein the so-called embryos are developed in a time range between 3-10 weeks.

32. A method according to claim 24, wherein the so-called embryos are developed in a time range between 5-8 weeks.

33. A method according to claim 24, wherein the ratio of ammonium to nitrate is 10:90.

34. A method according to claim 24, wherein the ratio of ammonium to nitrate is 20:80.

35. A method according to claim 24, wherein the ratio of ammonium to nitrate is 30:70.

36. A method according to claim 24, wherein the carbon source is maltose.

37. A method according to claim 24, wherein the concentration of maltose is 3% and 9%.

38. A method according to claim 24, wherein the maturation promoter is ABA or its analogues.

39. A method according to claim 24, wherein the concentration of ABA in said medium has a range between 60 to 100 uM.

40. A method according to claim 24, wherein the desiccating agent in said medium is "gellan gum".

41. A method according to claim 24, wherein the concentration of "gellan gum" in said medium is in a range of 0.5 to 0.65. CONSERVATION IN REFRIGERATION, CULTIVATION IN SUSPENSION AND MATURATION OF SOMATIC EMBRYOS OF GYMNOSPERMS ABSTRACT A new method to obtain mature somatic embryos of gymnosperms, based on refrigeration and suspension culture to conserve the embryogenic capacity of immature somatic embryos. Which consists of maintaining immature somatic embryos in a liquid medium at 4 ° C for a maximum period of one year; the reinduction and proliferation of immature embryos, treatment to reduce the proliferation and maturation of somatic embryos. With the exception of the last stage, everything was done by suspension cultures. The germination of the embryos and the development of the plant were done in a conventional manner.